The biological reduction of a colorless tetrazolium salt 1 to a colored formazan 2 is widely used to indicate and measure cell viability in cell cultures (1,2). ##STR1## Numerous laboratories have consequently used tetrazolium salts for many years in assays to distinguish living cells from dead ones. There are several characteristics which suit tetrazolium salts to this purpose.
Tetrazolium salts are reduced to the intensely colored formazans by the functioning electron transport systems of viable cells, either directly or through the intermediacy of electron carriers such as phenazine methosulfate or menadione. The direction of the color change, from the colorless oxidized form to a colored reduced form, is practically unique among dye-stuffs. Viable cells develop color in direct proportion to their metabolic activity. The colorless tetrazolium reagent is an ionized salt, soluble in water and capable of passing into cells. The colored reduction product, the formazan, is not ionic, is insoluble in water, and is deposited within viable cells in a manner well suited to histochemical evaluation. A number of different structural groups can be attached to the tetrazolium core to influence color, redox potential, solubility, and histochemical characteristics of the tetrazolium - formazan couples.
Over a thousand different tetrazolium compounds have been synthesized of which about a dozen are in common use for cell viability assays. Some of the procedures using tetrazolium salts currently in vogue have problems in that they are cumbersome, labor intensive, and ill-suited to volume throughput or automation. Typically, in such prior art procedures the formazan dye is deposited as an insoluble precipitate within cells of the culture which, in the standard procedure, must be spun down, mounted on a microscope slide, and evaluated (rather subjectively) by a skilled microscopist. Recently procedures have been described in which the cells are lysed with solvents such as DMF, DMSO, and 2-propanol, and the dye thereby brought into solution to be measured spectrophotometrically (3,4). These procedures give less subjective quantitation, but are no more convenient nor less labor intensive than the earlier methods.
One tetrazolium compound used recently for cell viability assays is the tetrazolium disulfonic acid XTT, a tetrazolium disulfonic acid wherein R.sub.1 =carbanilido, and R.sub.2 =R.sub.3 =2-methoxy-4-nitrobenzene5-sulfonic acid (5). An assay utilizing this compound capitalizes on the water solubility of the formazan disulfonic acid produced upon reduction of the tetrazolium zwitterion-salt. The dye diffuses out of the cells in which it is produced and into the culture medium. Optical density is read directly. The steps involving removal of the medium and extraction with solvent are obviated and the procedure becomes amenable to full automation.
There are problems with XTT however. The synthesis for XTT is problematic and the XTT formazan is yellow-orange in color (in water), leading to difficulties in the differentiation of the formazan from a generally yellow cell culture medium background. Problems also arise in certain cell types where XTT forms precipitates.
To be most useful in spectrophotometric assays, a formazan should be purple, blue or violet. XTT shows that there is an advantage in utilizing a tetrazolium compound which reduces to a water soluble formazan which is capable of diffusing out of the cells into the culture medium where it could be measured by standard spectrophotometers, and also the utility of sulfonate groups in achieving such water solubility in the formazans. The present invention provides these advantages while further providing structural flexibility allowing chemical modification to overcome many of the problems associated with XXT.